The physiological actions of serotonin (5-hydroxytryptamine) are mediated by 14 different receptor subtypes, all but one belonging to the class of G-protein coupled receptors (Hoyer et al., 2002, Pharmacol Biochem Behav, 71:533-554). These receptors are divided into seven distinct classes (5-HT1 to 5-HT7) largely on the basis of their structural and functional characteristics. The elucidation of the molecular, pharmacological, and physiological characterization of these receptors helps determine the roles of these receptors and their utilities as therapeutic targets.
The 5-HT2 receptor family comprises three receptor subtypes: 5-HT2A, 5-HT2B and 5-HT2C receptors (for a recent review, see Leysen et al., 2004, Curr Drug Target CNS Neurol Disord, 3:11-26). The 5-HT2 receptors are linked to the Gq family of G-proteins and subsequent activation of phospholipase C, induction of phosphoinositide metabolism and an increase in intracellular calcium concentration (Foguet, et al., 1992, Embo J, 11:3481-3487; Jerman et al., 2001, Eur J Pharmacol, 414:23-30; Porter, 1999, Br J Pharmacol, 128:13-20; Roth et al., 1998, Pharmacology & Therapeutics 79:231-257). Both 5-HT2A and 5-HT2B receptors are found in the central nervous system and periphery, whereas 5-HT2C is restricted to the central nervous system (Hoyer et al., 2002, Pharmacol Biochem Behav, 71:533-554; Leysen, J. E., 2004, Curr Drug Target CNS Neurol Disord 3:11-26).
The 5-HT2A receptors mediate contractile responses in vascular smooth muscle (Bhatnagar et al., 2004, J Biol Chem, 279 (33):34614-34623; Cohen et al., 1981, J Pharmacol Exp Ther, 218:421-425). In addition, platelet aggregation and increased capillary permeability following exposure to 5-HT have been attributed to 5-HT2A receptor-mediated functions (Bhatnagar et al., 2004, J Biol Chem, 279 (33):34614-34623; de Chaffoy de Courcelles at al., 1985, J Biol Chem 260:7603-7608; Hall et al., 2000, Synapse 38:421-431). Centrally, 5-HT2A receptors are principally located in cortex, claustrum, basal ganglia and in several brain stem nuclei (Fonseca et al., 2001, Brain Res Mol Brain Res, 89:11-19). 5-HT2A receptors in the medial nucleus of the tractus solitarius play a role in 5-HT-induced hypotension and bradycardia (Huang et al., 2003, J Comp Neurol, 455:270-280). 5-HT2A receptor activation also stimulates hormone secretion, e.g. ACTH, corticosterone, oxytocin, renin and prolactin secretion (Van de Kar et al., 2001, J Neurosci, 21:3572-3579). Moreover, 5-HT2A receptor agonists mediate certain behavioral, emotional, and cognitive syndromes and disorders (Bhatnagar et al., 2004, J Biol Chem, 279 (33):34614-34623). Head twitching in rat and mice can be inhibited with selective 5-HT2A antagonists (Schreiber et al., 1995, J Pharmacol Exp Ther, 273:101-112), and certain antipsychotics and antidepressant drugs are 5-HT2A antagonists (Roth et al., 1998, Pharmacology & Therapeutics 79:231-257).
Activation of the 5-HT2B receptor leads to fundic smooth muscle contraction (Foguet, et al., 1992, Embo J, 11:3481-3487; Kursar et al., 1992, Mol Pharmacol 42, 549-557). The 5-HT2B receptor is found throughout the human gastrointestinal tract where it mediates contractile responses. The 5-HT2B receptor has also been detected in discrete nuclei (Bonaventure et al., 2002, Brain Res 943:38-47; Duxon et al., 1997). Stimulation of 5-HT2B receptors on endothelial cells of the cerebral arteries by 5-HT causes release of nitric oxide, leading to vascular relaxation (Schmuck et al., 1996, Eur J Neurosci, 8:959-967). Hence, stimulation of 5-HT2B receptors on meningial blood vessels could be a trigger for migraine, perhaps explaining the reported prophylactic effect of 5-HT2 receptor antagonists (Schmuck et al., 1996, Eur J Neurosci, 8:959-967). 5-HT2B receptor activation may also be involved in the development of cardiac valvulopathy associated with norfenfluramine and other serotonergic medication (Fitzgerald et al., 1999, J Neurochem, 72:2127-2134).
The 5-HT2A and 5-HT2B receptor subtypes have been cloned from several species. The 5-HT2A receptors from hamster, human, monkey, mouse, pig, rat and sheep all have a similar length of 471 amino acids; the 5-HT2B receptors from human, mouse and rat have a length of 481, 504 and 460 amino acids, respectively (for review, see Kroeze et al., 2002, Curr Top Med Chem, 2:507-528). Each of the genes for the 5-HT2A and 5-HT2B receptors possesses three exons. Species differences in the binding of certain ligands between human and rat 5-HT2A receptors have been reported; for instance, ergolines appeared to display higher affinity for the rat than for the human receptor (Pazos et al., 1984, Eur J Pharmacol, 106:539-546). This is due to an amino acid variation (S242A) in the fifth transmembrane domain between the human (242S) and rat (242Ala) 5-HT2A receptor (Roth et al., 1998, Pharmacology & Therapeutics, 79:231-257). Additionally, differences in ligand binding between the human and rat 5-HT2B receptors have been reported, with ergolines and certain atypical antipsychotics displaying higher affinity for the human 5-HT2B receptor (Wainscott et al., 1996, J Pharmacol Exp Ther, 276:720-727).
The actions of 5-HT and 5-HT mimetics as they relate to many of the functions, syndromes, and disorders mentioned above have been studied in canine (e.g., see Bush, 1987, J Pharmacol Exp Ther, 240:674-682; Prins et al., 2001, Br J Pharmacol, 134:1351-1359; Shoji et al., 1990, Eur J Pharmacol, 190:247-250). The canine is also used to assess physiological liabilities, such as cardiovascular liabilities, of early drug candidates. There is a need to identify canine 5-HT2A and 5-HT2B receptors for use in interpreting data obtained from studies in canine, and in obtaining activity and binding affinity values for compounds in order to assay for ones that complex with or otherwise modulate canine 5-HT2A or 5-HT2B receptors.